Through a series of recent studies with gold nanoparticles (GNPs), the current research team has shown that x-ray fluorescence computed tomography (XFCT), traditionally a synchrotron-based imaging modality, can be performed on a benchtop setting for small-animal-sized objects using an ordinary diagnostic energy range polychromatic x-ray source. Consequently, one can now envision a widespread use of XFCT for biomedical research, especially in light of ever-growing interest in high atomic number (Z > ~50) metal nanoparticles such as GNPs for various cancer diagnostic/therapeutic applications and preclinical testing of such applications using small animal models. Once fully developed, benchtop XFCT will offer a powerful tool to determine the biodistribution of GNPs and other metal NP probes in vivo without cumbersome and time-consuming ex vivo analysis after sacrificing animals. Moreover, it will enable seamless multimodal imaging with micro-CT (CT) in a single platform, resulting in images showing greater anatomical details while retaining all the benefits from spectroscopic quantitative imaging. Furthermore, when performed with bioconjugated/functionalized GNPs and/or other metal NP probes, benchtop CT/XFCT will provide an unprecedented multimodal/multiplexed molecular imaging option for more effective drug discovery and development. Despite significant research advances on benchtop XFCT in recent years, however, there are some critical technical challenges that must be overcome to realize these possibilities. Thus, this project brings together a team of experts from Brookhaven National Laboratory, University of Massachusetts Medical School, and The University of Texas MD Anderson Cancer Center to surmount the key technical hurdles that prevent the envisioned benchtop CT/XFCT system from being used routinely for in vivo imaging under realistic constraints. The following three specific aims will be pursued to achieve the goal of this project: (1) Design and optimization of a benchtop CT/XFCT system; (2) Development of novel high energy-resolution solid state array detectors for benchtop XFCT; (3) Investigation of multimodal/multiplexed molecular imaging capability of a benchtop CT/XFCT system. Upon successful completion, a practical benchtop CT/XFCT system will become ready for routine preclinical molecular imaging studies and further improvements. A fully-developed benchtop CT/XFCT system will offer novel imaging options that can considerably improve the efficacy of preclinical testing of NP- based diagnostic/therapeutic strategies, thereby increasing the likelihood of bringing paradigm-shifting advancements in cancer nanomedicine. A novel high energy-resolution detector system developed from this project will also offer new opportunities for a wide range of quantitative x-ray imaging applications beyond benchtop XFCT.